Cells infected by two or more genetically different viruses can produce heterozygotic virions, from which recombinant viruses are generated. Therefore, double infection directly affects the overall recombination rate. We have studied the nature of HIV-1 double infection in cultured and primary T cells by direct cell-free virus infection and by cell-mediated virus transmission. We have observed that HIV-1 double infection occurs significantly more frequently than it would at random in both transmission pathways. This is the first study that reveals the nature of retroviral double infection. We have also dissected the mechanisms that cause nonrandom HIV-1 double infection, and demonstrated that preference in entry to the host cell played an important role in nonrandom double infection. We are currently establishing other experimental systems to continue our study of retroviral double infection, including examining clinical samples.